Heart disease is the single largest killer of Americans. Those that survive a heart attack have a higher risk of another attack, heart failure, and stroke. Consequently, there is a need to develop approaches for repairing the heart and improving the quality of life after a heart attack. The concept of regenerative medicine through the use of stem cells is gaining attention as a potential therapy for restoring cardiac function. Hurdles currently faced in the development of cellular therapies for the clinic are (1) the limited availability of cell surface markers for selecting pure cell populations and for tracking differentiation and (2) the lack of knowledge of how in vitro differentiation and in vivo development of cardiomyocytes correlate. The long term goal of this project is to characterize and understand stem cell differentiation. We aim to achieve this by identifying and characterizing specific cell surface protein 'barcodes' useful for marking specific cell types. The major objectives are to first address the need for more cell surface markers that are useful for characterizing specific cell types, stages, and lineages, and specifically, we will focus on pluripotent cells, cardiac progenitors, and ventricular cardiomyocytes. We will use state-of-the-art analytical biochemistry and mass spectrometry approaches to identify and quantify novel surface proteins in a model cell line and these proteins will then be evaluated for their utility in live cell sorting to obtain pure, defined populations. Subsequently, we will apply the knowledge gained from the in vitro work to understanding cardiac cell development in the animal. Specifically, we will determine whether the in vitro markers are also present at the appropriate time and space during embryonic development. The specific aims are designed to develop an understanding of the function of the cell surface markers and whether the molecular events during in vitro differentiation mimic those during in vivo development of cardiomyocytes. The supportive environment at my new institution will facilitate the continued success of this proposal by providing access to state-of-the art instrumentation, mentors with specialized expertise, and the forum to support discussion of clinically relevant research.